Investigation of ethyl alcohol and beta-carotene effect on two models of carcinogenesis

The effects of ethyl alcohol and synthetic beta-carotene have been studied on two models of carcinogenesis in mice BALB/c. Lung tumours were induced with organotropically acting urethane (given by i.p. injections, total dose--100 mg/mouse) subcutaneous tumours were induced with locally acting benzo(a)pyrene (single injection, 2 mg/mouse) beta-Carotene was given 3 times per week 0.4 mg/mouse by gastric intubations and 10% ethanol was given instead of drinking water until the end of experiments (4-6 months). Results showed that beta-carotene did not significantly inhibit lung adenomogenesis and may moderately delay subcutaneous tumours occurence. In ourstudies chronic ethanol intake did notshow significant influence on this delay.     

Evaluation of chromosomal aberrations in bone marrow of 1C3F1 mice

The chemical caprolactam (CAP) was tested for induction of chromosomal aberrations in bone marrow of male and female 1C3F1 mice at a single dose of 1000 mg/kg by oral intubation of an aqueous solution at a volume of 0.1 ml/10 g of body weight. Bone marrow was sampled from groups of 10 animals 24, 30 and 48 h after treatment. CAP did not produce chromosomal aberrations under the present experimental conditions. At the same time, benzo[a]pyrene, used as a positive control, significantly increased the aberration rates at a dose of 63 mg/kg.     

Inhibition of mitomycin C-induced sister-chromatid exchanges in mouse bone marrow cells by the immunopotentiators Krestin and Lentinan

We have carried out an investigation to determine whether or not the sister-chromatid exchange frequencies (SCEs) observed in bone marrow cells in mice treated with mitomycin C (MMC) are inhibited by the immunopotentiators Krestin and Lentinan. We found that mitomycin C (2 mg/kg, i.v.)-induced SCEs were inhibited in 27% of the mice treated with Krestin (300 mg/kg, i.p.) and in 23% of the mice treated with Lentinan (1 mg/kg, i.p.). The effects of Krestin were found to be dose-dependent in inhibition of MMC-induced SCEs while those of Lentinan were not. Our findings therefore suggest that Krestin and Lentinan are not only useful for cancer treatment as immunopotentiators in combination with anticancer drugs but may also prevent the increase of chromosomal damage induced by anticancer drugs.     

Prior bleeding enhances the sensitivity of the in vivo micronucleus test

It has been reported that the sensitivity of the in vivo mouse bone marrow micronucleus test can be increased by inducing erythropoiesis with exogenous erythropoietin prior to treatment (Suzuki et al., 1989). In these studies we demonstrate that removing approximately 0.5 ml of blood from an adult male BDF1 mouse, another method for increasing the rate of erythropoiesis, synergistically increased the frequency of bone marrow micronucleated polychromatic erythrocytes induced by mitomycin C, with maximal enhancement occurring when the mutagen was given 24 h after bleeding. This enhancement response was also demonstrated for benzo[a]pyrene and dimethylnitrosamine but not for 2-acetylaminofluorene. These results indicate that bleeding mice prior to chemical treatment is a simple method for increasing the sensitivity of the micronucleus assay.     

Importance of the route of administration for genetic differences in benzo[a]pyrene-induced in utero toxicity and teratogenicity

C57BL/6N (Ahb/Ahb) mice have a high-affinity Ah receptor in tissues, whereas AKR/J and DBA/2N (Ahd/Ahd) mice have a poor-affinity Ah receptor. The cytochrome P1-450 induction response (enhanced benzo[a]pyrene metabolism) occurs much more readily in Ahb/Ahb and Ahb/Ahd than in Ahd/Ahd mice, at any given dose of the inducer benzo[a]pyrene. Embryos from the AKR/J X (C57BL/6N)(AKR/J)F1 and the reciprocal backcross were studied during benzo[a]pyrene feeding of the pregnant females. Oral benzo[a]pyrene (120 mg/kg/day) given to pregnant Ahd/Ahd mice between gestational day 2 and 10 produces more intrauterine toxicity and malformations in Ahd/Ahd than Ahb/Ahd embryos. This striking allelic difference is not seen in pregnant Ahb/Ahd mice receiving oral benzo[a]pyrene. Pharmacokinetics studies with [3H]benzo[a]pyrene in the diet and high-performance liquid chromatographic analysis of benzo[a]pyrene metabolism in vitro by the maternal intestine, liver, and ovary and the embryos of control and oral benzo[a]pyrene-treated pregnant females are consistent with "first-pass elimination" kinetics and differences in benzo[a]pyrene metabolism by the embryos and/or placentas versus maternal tissues. In the pregnant Ahd/Ahd mouse receiving oral benzo[a]pyrene, little induction of benzo[a]pyrene metabolism occurs in her intestine and liver; this leads to much larger amounts of benzo[a]pyrene reaching her embryos, and genetic differences in toxicity and teratogenesis are manifest. In the pregnant Ahb/Ahd mouse receiving oral benzo[a]pyrene, benzo[a]pyrene metabolism is greatly enhanced in her intestine and liver; this leads to less benzo[a]pyrene reaching her embryos, much less intrauterine toxicity and malformations, and no genetic differences are manifest. More toxic metabolites (especially benzo[a]pyrene 1,6- and 3,6-quinones) are shown to occur in Ahd/Ahd embryos than in Ahb/Ahd embryos. In additional studies, no prenatal or neonatal "imprinting" effect in C57BL/6N mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 on benzo[a]pyrene metabolism later in life was detectable. These genetic differences in intrauterine toxicity and teratogenicity induced by oral benzo[a]pyrene are just opposite those induced by intraperitoneal benzo[a]pyrene [Shum et al., '79; Hoshino et al., '81). The data in the present report emphasize the importance of the route of administration when the teratogen induces its own metabolism.     

Chemoprotective effects of carnosine against genotoxicity induced by cyclophosphamide in mice bone marrow cells

The protective effects of carnosine as a natural dipeptide were investigated in mouse bone marrow cells against genotoxicity induced by cyclophosphamide. Mice were injected with solutions of carnosine at three different doses (10, 50 and 100?mg kg(-1) bw) for five consecutive days. On the fifth day of treatment, mice were injected cyclophosphamide and killed after 24?h. The frequency of micronuclei in polychromatic erythrocytes and the ratio of polychromatic erythrocyte/polychromatic erythrocyte?+?normochromatic erythrocyte [PCE/(PCE?+?NCE)] were evaluated by May-Grunwald/Giemsa staining. Histopathology of bone marrow was examined in mice treated with cyclophosphamide and carnosine. Carnosine significantly reduced micronucleated polychromatic erythrocytes (MnPCEs) induced by cyclophosphamide at all three doses. Carnosine at dose of 100?mg kg(-1) bw reduced MnPCEs 3.76-fold and completely normalized the PCE/(PCE?+?NCE) ratio. Administration of carnosine inhibited bone marrow toxicity induced by cyclophosphamide. It appeared that carnosine with protective activity reduced the oxidative stress and genotoxicity induced by cyclophosphamide in bone marrow cells of mice. Copyright ? 2012 John Wiley & Sons, Ltd.     

Induction of sister-chromatid exchange (SCE) and cell-cycle inhibition in mouse peripheral blood B lymphocytes exposed to mutagenic carcinogens in vivo

To determine the sensitivity of the mouse peripheral blood lymphocyte (PBL) culture system, male B6C3f1 mice were injected i.p. with either 2-acetylaminofluorene (AAF) (20, 40, 80, 160 mg/kg), benzo[a]pyrene (BP) 25, 75, 150, 300 mg/kg), dichlorvos (DCV) (5, 15, 25, 35 mg/kg), ethyl methanesulfonate (EMS) (10, 30, 90, 180, 270 mg/kg), or N-nitrosomorpholine (NM) (37.5, 75, 150, 300 mg/kg) dissolved in either RPMI 1640 (DCV, EMS, NM) or sunflower oil (AAF, BP). 24 h later blood was removed by cardiac puncture, and the lymphocytes were cultured in the presence of lipopolysaccharide for analysis of SCE in B lymphocytes. All 4 mutagenic carcinogens (AAF, BP, EMS, NM) induced significant dose-related increases in SCE frequency. DCV, a potent neurotoxicant, caused no change in the baseline SCE frequency. At the highest concentration of each chemical examined, AAF caused a 1.6-fold increase, EMS a 1.8-fold increase, NM a 3.0-fold increase, and BP a 3.1-fold increase in SCE frequency compared to concurrent controls. A comparison of these results for PBLs with those reported in the literature for bone marrow cells indicates that PBLs offer a good quantitative and qualitative estimate of the SCE-inducing potential for these 5 compounds in bone marrow cells.     

Cytogenetic effect of mitomycin C and cytosine arabinoside on mice of different genotypes]

Cytogenetic effect of mitomycin C (MC) and cytosine arabinoside (CA) on bone marrow cells of male mice of the strains 101/HY, C57BL/6Y C,3H/SnY and of the (C3HX101) F1 hybrids was studied. The frequencies of cells with chromosome aberrations after the treatment with MC at a 5 mg/kg dose were 54,4%; 41,8%; 40,4% and 26,8% in 101H, B6, C3H/Sn mice and in the F1 hybrids (C3HX101) respectively. The frequencies of cells with chromosome aberrations after the treatment with CA at a 500 mg/kg dose were 25,2%; 17,8%; 10,8% and the 101/H, B6, C3H/Sn mice and in the F1 hybrids (C3HX101) respectively. Both mutagens induced the greatest number of chromosome aberrations in the 101/H strain and the smallest number in the F1 hybrid (C3HX101). A positive correlation was established between the levels of induced and spontaneous chromosome lesions.     

Differences in murine procarcinogen activation enzymes are not accompanied by parallel differences in procarcinogen-induced sister-chromatid exchange

C57B1/6 and DBA/2 mice, strains in which there is marked induction of hepatic monooxygenase activity by phenobarbital, were tested for in vivo sister-chromatid exchange (SCE) formation in response to cyclophosphamide, an agent metabolized by this inducible enzyme system. Baseline SCE frequencies were between 4 and 6 SCEs/cell in regerating liver and bone marrow of both strains of mice. Administration of cyclophosphamide (5 mg/kg) led to an increase of nearly 8 SCEs/cell in both tissues of C57B1/6 mice and an increase of more than 10 SCEs/cell in DBA/2 mice. Prior exposure to phenobarbital induced p-chloromethylaniline demethylase activity in regenerating liver of both mouse strains approx. 6-fold, but the changes in measured SCE frequencies were not significantly different from those obtained in the absence of enzyme induction. These results, together with our previous observation that induction of by 3-methylcholanthrene of benzo[a]pyrene hydroxylase activity in the same mouse strains was not accompanied by a comparable change in benzo[a]pyrene-induced SCE formation, reinforce the impression that simple assays of differences in mixed function oxidase activities may not necessarily be good predictors of hereditary differences in the response to genetic damage by procarcinogens which are presumed to be metabolized by these enzymes.     

Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice

Nicotne has been tested in the conventional mouse bone marrow assay. Single doses of 1 mg/kg bw or 2 mg/kg bw were given by oral intubations and bone marrow was sampled at 24 h (1 mg/kg) or at 6, 12 and 18 h after treatment (2 mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1 mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6 h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nocotine is not clastogenic at the doses and time intervals tested in the present experiments.     

Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice

Nicotine has been tested in the conventional mouse bone marrow assay. Single doses of 1mg/kg bw or 2mg/kg bw were given by oral intubations and bone marrow was sampled at 24h (1mg/kg) or at 6, 12 and 18 h after treatment (2mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nicotine is not clastogenic at the doses and time intervals tested in the present experiments.     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.     

Relation of the antimutagenic activity of simple phenols to the number of hydroxyl groups

The influence of mono-phenol, di-resorcinol and tri-pyrogallol hydroxyl groups of simple unsubstituted phenols on the mutagenic potentials of benzo(a)pyrene was studied in vivo (micronuclear test on bone marrow polychromatic erythrocytes) and in vitro (test of direct point mutations at V79/HGPRT system induced by metabolic activation by mouse liver microsomal enzymes). The phenols decreased the mutagenic activity of benzo(a)pyrene in in vivo tests, with pyrogallol being the most active, it followed by resorcinol and phenol. The mixtures of benzo(a) pyrene + pyrogallol and benzo(a)pyrene + resorcinol were significantly less mutagenic in in vitro tests than benzo(a)pyrene and benzo(a)pyrene + phenol.     

Potentiation by caffeine of the frequencies of micronuclei induced by mitomycin C and cyclophosphamide in young mice

Employing the micronucleus test in mouse bone marrow and in fetal mouse liver, the possible clastogenicity of caffeine as well as its influence on MMC- and CP-induced micronucleus levels were studied. The treatment of male and female C57Bl or BDF1 (C57Bl x DBA2) mice with caffeine (1 or 3 x 50 mg/kg and 100 mg/kg, s.c.) had no clastogenic effect in mouse bone marrow or in the fetal livers and maternal bone marrow when pregnant mice were injected with caffeine on day 16-17 of gestation. MMC (2.0 mg/kg, i.p.) increased up to 10-30-fold the number of MNPCEs in bone marrow compared to a 3-7 fold elevation of MNPCEs in fetal liver. A similar effect was also established in pregnant mice treated with CP (30 mg/kg, i.p.). No significant sex differences in spontaneous and MMC- or CP-induced MNPCEs levels were established in C57Bl and BDF1 mice. However, a significantly higher spontaneous rate of MNPCEs as well as a better-expressed responsiveness to the clastogenic activity of MMC and CP were established in C57Bl compared to BDF1 mice. The pregnancy had no effect on MMC- or CP-induced clastogenicity although a tendency to a decreased sensitivity to the damaging activity of MMC seemed to be detected in pregnant C57Bl mice compared to virgin female animals. The combined treatment of mice with caffeine (3 x 100 mg/kg) and MMC or CP caused an up to 45-49% potentiation of clastogenesis in the bone marrow of male, female and pregnant female C57Bl and BDF1 mice but not in fetal mouse livers.     

Sampling times in micronucleus testing.

A series of micronucleus inducers were evaluated in the mouse bone marrow micronucleus test to determine if a 72-h sampling time enhances the sensitivity for detecting genotoxic agents. Male and female Swiss albino mice were dosed once with 7,12- dimethylbenz[a]anthracene, 6-mercaptopurine, benzo[a]pyrene, benzene, cyclophosphamide, 2-acetylaminofluorene, tubulazole, or mitomycin C. According to the EEC and OECD guidelines, the mice were killed at 24, 48 and 72 h after dosing. All test compounds induced an increase in the number of micronucleated polychromatic erythrocytes at 24 and/or 48 h. From the results obtained, it was evident that the 72-h sampling time does not enhance the sensitivity of the micronucleus test. The present data show that for screening purposes two sampling times at 24 and 48 h are sufficient to detect clastogens as well as aneugens. Although quantitative differences were found in sensitivity to micronucleus inducers between male and female mice, no qualitative differences were observed between the two sexes.     

Inhibition of clastogenicity of benzo[a]pyrene and of its trans-7,8-dihydrodiol in mice in vivo by fruits,vegetables, and flavonoids

In the in vivo mouse bone marrow micronucleus assay, homogenates of spinach, artichoke, peaches, and blue grapes as well as commercial concentrates of these vegetables and fruits reduced induction of micronuclei by benzo[a]pyrene (BaP) by 43-50%. Concentrates of strawberries (31% reduction) and of cauliflower (20% reduction) were less potent. Inhibition of genotoxicity by spinach and peaches was not caused by any delay in maturation of micronucleated erythrocytes as shown by experiments with sampling times of 24, 48, and 72 h after dosing of BaP. Pre-treatment of the mice with spinach 48, 24, and 12h before application of BaP resulted in a 44% reduction of micronuclei while peaches generated only a marginal effect. A post-treatment procedure administering spinach or peaches 6h after dosing of BaP did not indicate any protective effects. When trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-OH) was applied for induction of micronuclei spinach and peaches reduced the number of micronuclei by 55 and 48%, respectively. Pre-treatment of mice with spinach 96, 72, and 60 h before sacrifice caused a decline of hepatic 7-ethoxyresorufin-O-dealkylase (EROD) and of 7-pentoxyresorufin-O-dealkylase (PROD) activities by factors of 2.2 and 1.4, respectively. However, statistical significance was not reached. On the other hand, peaches had no influence on hepatic EROD or PROD activities. The flavonoids quercetin and its glucoside isoquercitrin, administered orally in doses of 0.03 mmol/kg body weight simultaneously with intraperitoneally given BaP, reduced the number of micronuclei in polychromatic erythrocytes of the bone marrow of mice by 73 and 33%. Ten-fold higher concentrations, however, reversed the effects with a particular strong increase observed with isoquercitrin (+109%; quercetin: +16%).     

Inhibitory effects of coffee on the genotoxicity of carcinogens in mice

The mouse bone marrow micronucleus test was carried out to evaluate the possible inhibitory effects of 3 doses (125, 250 and 500 mg/kg) of standard instant coffee on the in vivo genotoxicity of 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP), aflatoxin B1 (AFB1) and urethane (UR). Coffee was orally administered twice, 2 and 20 h before the carcinogens were injected intraperitoneally. From the results obtained, it was evident that the administration of 250 and 500 mg coffee/kg body weight could significantly inhibit the in vivo genotoxicity of these carcinogens. A linear dose response was observed for the inhibitory effect of coffee. Furthermore, inhibition of genotoxicity by coffee was observed in bone marrow cells which were sampled at 6-h intervals (48, 54, 60, 66 and 72 h) from the time of peak induction of micronuclei by DMBA.     

Acceleration of Recovery from Mitomycin C-induced Leukopenia by FK-565

FK-565 restored the number of granulocytes in the bone marrow in mice treated with mitomycin C (MMC). Granulocyte counts recovered to the normal level 3 days after MMC treatment at a dose of 0.1 mg/kg of FK-565. The restorative activity of FK-565 was more effective than that of muramyldipeptide (MDP) or lipopolysaccharide (LPS).     

Stabilization of Membrane Bound Enzyme Profiles and Lipid Peroxidation by Withania Somnifera Along with Paclitaxel on Benzo(a)pyrene Induced Experimental Lung Cancer

The present study was aimed to evaluate the therapeutic effects of Withania somnifera along with paclitaxel on lung tumor induced by benzo(a)pyrene in male Swiss albino mice. The levels of ATPase enzymes and lipid peroxidation were evaluated in lung cancer bearing mice, in erythrocyte membrane and tissues. The extent of peroxidation was estimated by measuring the thiobarbituric acid-reactive substances. Simultaneously the activities of different ATPases (Na⁺/K⁺-ATPases, Mg²⁺-ATPases and Ca²⁺-ATPases) were determined. The alterations of these enzyme activities in membrane and tissues were indicative of the tumor formation caused by benzo(a)pyrene (50 mg/kg body weight, orally) in cancer bearing animals. The activities of these enzymes were reversed to near normal control values in animals treated with Withania somnifera (400 mg/kg b.wt, orally) along with paclitaxel (33 mg/kg b.wt, i.p). Treatment with Withania somnifera along with paclitaxel altered these damage mediated through free radicals, and the treatment displays the protective role of these drugs by inhibiting free radical mediated cellular damages. Over, based on the data providing a correlation Withania somnifera along with paclitaxel provide stabilization of membrane bound enzyme profiles and decreased lipid peroxidation against benzo(a)pyrene induced lung cancer in mice.     

Correction of a T-immunodeficit in mice by bone marrow transplantation from donors treated with hydrocortisone]

Adult CBA mice were exposed to thymectomy, lethal irradiation, and protection by syngeneic bone marrow transplantation. In some experiments syngeneic bone marrow of donors, treated with hydrocortisone in a dose of 125 mg/kg for 3 days was used. The bone marrow of these donors contained cells with the Q-marker. Thymectomized and lethally irradiated animals subjected to the transplantation of syngeneic bone marrow from hydrocortisone-treated donors rejected the skin allotransplants, and the lymph node cells of these mice suppressed the endogenous colony-formation in the sublethally-irradiated hybrids (CBA X C57Bl/6) F1.